WO2022001189A1 - 一组gitr单克隆抗体及其医药用途 - Google Patents

一组gitr单克隆抗体及其医药用途 Download PDF

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WO2022001189A1
WO2022001189A1 PCT/CN2021/080981 CN2021080981W WO2022001189A1 WO 2022001189 A1 WO2022001189 A1 WO 2022001189A1 CN 2021080981 W CN2021080981 W CN 2021080981W WO 2022001189 A1 WO2022001189 A1 WO 2022001189A1
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gitr
seq
nos
amino acid
humanized
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邱均专
孙自勇
王振生
孙锴
周漫
陈均勇
孙键
区日山
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东大生物技术(苏州)有限公司
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Priority to EP21831586.9A priority patent/EP4212549A1/en
Priority to US18/013,504 priority patent/US20230295323A1/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/75—Agonist effect on antigen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention belongs to the field of tumor and immunology drugs, and in particular, the present invention relates to a group of anti-GITR monoclonal antibodies and their medical uses.
  • GITR is a glucocorticoid-induced TNFR-related protein, also known as TNFRSF18 or CD357; it is a type I transmembrane protein that is expressed at high levels on Treg cells and at low levels on naive and memory T cells [Shimizu et al, (2002), Nat. Immunol. 3: 135-42; Nocentini et al, (2009), Adv. Exp. Med. Biol. 647: 156-73; McHughet et al, (2002), Immunity 16: 311 -23; Nocentini et al., (2005), Eur J Immunol 35: 1016-1022].
  • GITR GITR is also expressed at low to moderate levels on innate immune cells such as natural killer cells, macrophages and dendritic cells (DCs) [Clouthier et al., (2014), Growth Factor Rev. 25:91-106].
  • the ligand of GITR is a type II transmembrane protein belonging to the TNF superfamily, which is expressed in endothelial cells including B cells, macrophages and dendritic cells (DCs) as well as activated antigen-presenting cells (APCs) A certain amount of expression was observed on the cell surface of the 15059-15064].
  • GITR can be stimulated to enhance TCR-induced T cell proliferation and cytokine production through the interaction of GITRL or GITR agonist antibodies with GITR [Ronchetti et al., (2004), Eur. J. Immunol. 34:613-622 Tone et al., (2003), Proc.Natl.Acad.Sci.USA 100:15059-15064; Stephens et al., (2004), J. Immunol. susceptibility to induced immunosuppression [Ronchetti et al, (2007), Eur. J. Immunol. 37: 1165-1169; Stephens et al, (2004), J. Immunol. 173: 5008-5020].
  • GITR TNFR-associated factors 2 (TRAF 2) and 5 (TRAF5) and induces activation of transcription factors NF- ⁇ B and MAP kinase-induced signaling pathways [Snellet et al., (2011), Immunol. Rev. 244 [ Ronchetti et al, (2004), Eur. J. Immunol. 34: 613-622; Esparza et al, (2006), J. Biol. Chem. 281: 8555-64]. Activation of GITR can further lead to NFkB-dependent activation of TRAF6 and production of IL-9, which enhances DC cell function and activates the cytotoxic response of T lymphocytes [Kim et al., (2015), Nat. Med.
  • GITR signaling can also promote the degradation of FoxP3 [Cohenet et al, (2010), PLoS ONE 5:10436; Schaer et al, (2013), Cancer Immunology Research 1:320-331]. and phosphorylation of c-Jun N-terminal kinase (JNK) [Joetham et al., (2012), J Biol. Chem. 287: 17100-17108]; thereby, further inhibiting the suppressive function of Treg cells on immunity.
  • JNK c-Jun N-terminal kinase
  • anti-GITR antibodies have also been reported to reverse the inhibitory effect of Treg cells on B cell-mediated antibody production [Cava et al., (2004), J. Immunol. 173:3542-3548].
  • the GITR-activating antibody When the GITR-activating antibody is used alone, it can reduce the entry of Treg cells into tumor tissue and make Treg cells in the tumor lose the expression of foxp3; thereby locally eliminating the immunosuppressive effect of Treg cells in the tumor. Ultimately, in the tumor microenvironment, the Teff/Treg cell ratio is elevated and enhances Teff cell activity and function (Cohen et al., (2010), PLoS ONE 5:e10436). In ovarian cancer models, the combination of anti-GITR and anti-PD-1 antibodies improved overall patient survival; after 90 days of treatment, the tumor-free mice were 20 percent.
  • TILs tumor-entering leukocytes
  • the present invention obtains a group of anti-GITR monoclonal antibodies which have the activity of activating GITR and immune cells, and can block the interaction between GITR-L and GITR through hybridoma technology; Antibodies were humanized.
  • the antibodies have significant application prospects in the preparation of drugs for stimulating and enhancing immune responses and treating cancer-related drugs.
  • the present invention provides a group of anti-GITR monoclonal antibodies or antigen-binding fragments thereof, including heavy chain and light chain, characterized in that the amino acid sequence of CDR1 of the heavy chain is selected from SEQ ID NOs: 2, 10, 18, 26 , one of 34,42,50,58,66,74,82,90,98 and 106; the amino acid sequence of the CDR2 of the heavy chain is selected from SEQ ID NO:3,11,19,27,35, one of 43, 51, 59, 67, 75, 83, 91, 99 and 107; the amino acid sequence of the CDR3 of the heavy chain is selected from SEQ ID NOs: 4, 12, 20, 28, 36, 44, 52 , one of 60,68,76,84,92,100 and 108; the CDR1 amino acid sequence of the light chain is selected from SEQ ID NOs: 6,14,22,30,38,46,54,62,70,78 , one of 86, 94, 102 and 110; the amino acid sequence of the CDR
  • the present invention discloses the above-mentioned group of GITR monoclonal antibodies or antigen-binding fragments thereof, characterized in that the amino acid sequence of the variable region of the heavy chain is selected from SEQ ID NOs: 1, 9, 17, 25, 33 , one of 41, 49, 57, 65, 73, 81, 89, 97 and 105; the amino acid sequence of the light chain variable region is selected from SEQ ID NOs: 5, 13, 21, 29, 37, 45 , one of 53, 61, 69, 77, 85, 93, 101 and 109.
  • the present invention discloses humanization transformation of the heavy chain and light chain of the above-mentioned anti-GITR monoclonal antibody or its antigen-binding fragment, and the amino acid sequence of the humanized heavy chain variable region is selected from SEQ ID NO: one of 113, 115, 117 and 119; the amino acid sequence of the humanized light chain variable region is selected from one of SEQ ID NO: 114, 116, 118 and 120.
  • the present invention discloses a group of anti-GITR monoclonal antibodies or antigen-binding fragments thereof that have undergone humanization transformation, and the full-length amino acids of the humanized transformation heavy chain are selected from SEQ ID NOs: 121, 123, 125, One of 127, 128, 129 and 130; the full-length amino acid of the humanized engineered light chain is selected from one of SEQ ID NOs: 122, 124, 126 and 131.
  • the present invention correspondingly discloses the nucleotide sequence encoding the above-mentioned GITR monoclonal antibody or its antigen-binding fragment.
  • the present invention discloses the use of the above-mentioned anti-GITR fragments in the preparation of the following medicines: medicines for activating T lymphocytes, medicines for blocking the combination of GITR-L and GITR, or improving IL-2 and IFN- ⁇ in T lymphocytes. expressed drug.
  • the present invention discloses a monoclonal antibody conjugate, comprising a monoclonal antibody and a coupling part, wherein the monoclonal antibody is the GITR monoclonal antibody according to any one of claims 1-4 or its Antigen-binding fragments, the coupling moieties are one or more selected from radionuclides, drugs, toxins, cytokines, cytokine receptor fragments, enzymes, fluorescein, and biotin.
  • the present invention discloses the use of the above-mentioned monoclonal antibody conjugates in the preparation of the following medicines: medicines for activating T lymphocytes, medicines for blocking the combination of GITR-L and GITR, or for increasing the levels of IL-2 and IL-2 in T lymphocytes. Drugs that express IFN- ⁇ .
  • the present invention discloses the use of the above-mentioned monoclonal antibody conjugate in the preparation of a medicament for preventing and/or treating and/or adjuvant therapy of tumors.
  • the progress of the present invention is as follows: the present invention adopts a mammalian cell expression system to prepare recombinant GITR as an antigen to immunize mice, and fuses mouse spleen cells with myeloma cells to obtain hybridoma cells. After several times of cloning and screening of a large number of hybridoma cells, some monoclonal hybridoma cell lines were obtained.
  • hybridoma cell lines can produce monoclonal antibodies that specifically bind to GITR with high affinity, can promote the secretion of IL-2 and INF- ⁇ in mixed lymphocyte reaction, and can effectively block the binding of GITR-L to GITR, and
  • the genes encoding antibody light chain and heavy chain variable regions were cloned by RT-PCR, and humanized antibodies were constructed by complementary determinant grafting method.
  • humanized GITR antibodies can specifically bind to GITR protein with high affinity, can significantly promote T cells to secrete cytokines IL-2 and INF- ⁇ , and can block the binding of GITR-L to GITR.
  • the monoclonal antibody or its antigen-binding fragment of the present invention or the monoclonal antibody conjugate comprising the monoclonal antibody or its antigen-binding fragment of the present invention, can be used in the preparation of drugs for activating T lymphocytes, increasing T lymphocytes
  • Drugs that express IL-2 and INF- ⁇ in lymphocytes, drugs that block the combination of GITR-L and GITR, and drugs that prevent and treat or adjuvant tumors have good application prospects.
  • Fig. 1 is the EC50 of humanized anti-GITR antibody binding to GITR-mFc protein detected by ELISA method
  • Figure 2 shows the FACS test results of humanized GITR antibody binding to GITR on cell membranes
  • Figure 3 shows the blocking effect of humanized GITR antibody on the binding of GITR-L to Jurkat-GITR cells
  • Figure 4 shows the GITR activating activity of humanized anti-GITR antibodies based on the 293T-GITR-NFkb-luc cell reporter gene assay
  • Figure 5 shows the results of activating Jurkat-GITR cells to release cytokine IL2 by humanized GITR antibody without cross-linking
  • Figure 6 shows the result of activation of Jurkat-GITR cells to release cytokine IL2 by humanized GITR antibody in the presence of cross-linking
  • Figure 7 shows the effect of humanized GITR antibody on the production of cytokine IL2 by CD4 T cells
  • Figure 8 shows the effect of humanized GITR antibodies on the production of the cytokine IFN- ⁇ by CD4 T cells.
  • the purified human GITR(ECD)-mFc (Kim et al., 2007, Oncol Rep. 18:1189) was expressed and purified by Dongda Biotechnology.
  • the fusion protein was used as the antigen, with an equal volume of complete Freund's adjuvant (Sigma, Cat No: F5581) was fully emulsified, 6-8 week old Balb/c mice (purchased from Zhaoyan (Suzhou) New Drug Research Center Co., Ltd.) were subcutaneously immunized with an antigen immunization amount of 50 ⁇ g/mice; After the dose of antigen was fully emulsified with incomplete Freund's adjuvant (Sigma, Cat No: F5506), mice were subcutaneously immunized three times.
  • mice After three immunizations, the serum titers of the mice were determined, and booster immunization was performed intraperitoneally 3 days before fusion.
  • PEG Hybri-Max Sigma, Cat No: 7181
  • mouse spleen cells and SP2/0 cells were mixed at a ratio of 4:1.
  • the fused cells were added to 96-well plates (1 ⁇ 10 5 cells/well) containing 0.1 mL of 1 ⁇ HAT medium (Invitrogen, Cat No: 21060-017) per well.
  • the purpose of these tests is to screen out polyclonal hybridomas that can bind to GITR, block the interaction between GITR-L and GITR, and can significantly activate the activity of GITR molecules, and select polyclonal hybridomas with high binding and stimulatory activities.
  • the tumor is subcloned to obtain the desired monoclonal hybridoma antibody.
  • hybridoma cells were firstly subjected to ELISA and FACS assays to find and screen positive hybridomas that could bind to GITR, and then initially used
  • the 293T luciferase reporter gene assay screens polyclonal hybridomas with functional responses. A total of forty polyclonal hybridoma antibodies with strong binding to GITR were identified in this work. After the reporter gene test, the polyclonal hybridomas with significant binding and stimulating activity were selected for the next subclonal screening. Hybridoma cells with stronger binding to GITR were cryopreserved in liquid nitrogen.
  • FACS analysis of antibody binding capacity 50 ⁇ L of the hybridoma supernatant that was positive in the above ELISA assay was mixed with 50 ⁇ L of 293T-GITR cells (1 ⁇ 10 5 cells/well), added to a U-bottom 96-well plate and incubated for 1 hour , centrifuged and washed twice with FACS buffer (PBS+3% FCS), then added 1:400-fold diluted PE-labeled goat anti-mouse secondary antibody (Biolegend, Cat No: 405307), incubated for 30 minutes, and washed with FACS buffer. , the PE signal of 293T-GITR cells was detected by BD C6 flow cytometer.
  • FACS analysis for antibody blocking FACS analysis was performed using BD C6 to determine the blocking of biotinylated human GITR ligand binding to Jurkat-hGITR cells by hybridoma antibodies.
  • Jurkat-hGITR cells were suspended in FACS buffer (3% FBS in PBS).
  • Diluted hybridoma antibodies were added to the cell suspension and incubated in 96-well plates for 1 hour at 4°C.
  • Biotin-labeled human GITR ligand protein was added to the wells of a 96-well plate and incubated at 4 °C for 1 h. The incubated 96-well plate was washed 3 times and mouse anti-biotin PE antibody (Biolgend, cat. no. 409004) was added and incubated at 4°C for 0.5 hours.
  • Cells in 96-well plates were washed 3 times and finally analyzed with BD C6.
  • subcloning The purpose of subcloning is to establish monoclonal hybridoma cells to obtain monoclonal antibody clones.
  • Our subcloning method is based on a limiting dilution procedure to obtain hybridoma cells in a 96-well plate that yield a single clone in a single well. After multiple rounds (3 rounds) of subcloning, it was ensured that the resulting single clones were free of other cellular components. For each round of subcloning, ELISA, FACS, and reporter gene activation activity were detected.
  • a total of 14 anti-GITR monoclonal hybridomas were cultured and antibody purified, including 4E1, 8H4, 4E4, 3F5, 4C1, 7D7, 7E11, 10E11, 3G12, 4B11, 5F1, 5F6, 13G12, and 9B3.
  • Dulbecco's Modified Eagle's medium containing 10% hybridoma cells are fetal calf serum, 1% penicillin / streptomycin, 2% L- glutamine and 1% NaHCO 3 solution adjusting (GIBCO; Invitrogen Corporation, Carlsbad, CA) cultivated in.
  • Selected hybridoma cells were then adapted to serum-free medium and antibodies were purified from the supernatant using Protein-G columns (GE Healthcare). After washing with PBS, bound antibody was eluted with 0.1M glycine pH 3.0, then pH neutralized with 2.0M Tris.
  • Ultra-15 centrifugal concentrators (Amicon) were used for buffer exchange and antibody concentration.
  • Test analysis based on ELISA and FACS methods the methods used are similar to those described in Example 2 above, except that the test here uses purified antibodies at measured concentrations. Using serial dilutions of antibody concentrations and the corresponding results, one can calculate EC50 and IC50 values were obtained. Tables 4-7 show the test results for 14 antibodies. In addition, analysis of the cross-binding properties with monkey-derived GITR showed that the selected 14 antibodies had cross-binding reactions with monkey-derived GITR.
  • IL2 cytokine release from Jurkat-GITR cells was used as one of the indicators for evaluating the activation of GITR signaling pathway.
  • Jurkat-GITR cell cultures were performed in RPMI 1640 supplemented with 10% FBS.
  • 96-well plates were incubated with goat anti-human IgG (Jackson, catalog number: 109-005-008) and goat anti-mouse IgG (Jackson, catalog number: 115-006-071 ) at room temperature at room temperature Wrap overnight. After washing the plate wells, add 200 ⁇ L of blocking buffer and incubate for 1 hour at 37°C for blocking.
  • Human CD4 T cells were further purified on the basis of human-derived PBMC using a negative selection purification kit for CD4 T cells and RPMI 1640 medium supplemented with 10% FBS.
  • 96-well plates were incubated with goat anti-human IgG (Jackson, catalog number: 109-005-008 ) and goat anti-mouse IgG (Jackson, catalog number: 115-006-071 ) at room temperature Coat overnight at room temperature.
  • Table 9 Activation activity of hybridoma anti-GITR antibodies on immune cells based on detection of IL-2 and IFNg release from primary CD4 T cells.
  • the cloning analysis of the variable region gene of GITR antibody includes the following steps: using TRIzon (Cwbiotech, Cat No: CW0580) to lyse the GITR monoclonal hybridoma cell line, and extract the total RNA of the hybridoma cells.
  • RNA from hybridoma cells was reverse transcribed into cDNA using the HiFi Script cDNA synthesis kit (Cwbiotech, Cat No: CW2569).
  • degenerate primers were used to amplify the heavyweight of the antibody by PCR method (Kettleborough et al. (1993) Eur. J Immunology 23:206-211; Strebe et al. (2010) Antibody Engineering 1:3-14).
  • reaction mixture was extended by incubation at 72°C for an additional 7 minutes.
  • the PCR mixture was electrophoresed in a 1% agarose/Tris-borate gel containing 0.5 ⁇ g/ml ethidium bromide. DNA fragments of the expected size (about 400 bp for heavy and light chains) were excised from the gel and purified. 3 ⁇ l of the purified PCR product was cloned into pMD-18T vector (Takara, CAT#: D101A), transformed into DH5 ⁇ competent cells, plated and cultured at 37°C overnight. The single clone was picked from the culture plate, and the plasmid was extracted after expanding the culture, and the gene sequence of the antibody was determined. Based on the gene sequence of the antibody, its complementarity determinants (CDRs) and framework regions were analyzed.
  • CDRs complementarity determinants
  • the amino acid sequences of the CDR regions of the heavy and light chains of the antibody are shown in Table 1 and Table 2; the full-length amino acid sequences of the variable regions of the heavy and light chains of the antibody are shown in Table 3.
  • Amplified from the corresponding hybridoma clones were the variable region sequences of 14 antibodies (see Table): 4E1, 8H4, 4E4, 3F5, 4C1, 7D7, 7E11, 10E11, 3G12, 4B11, 5F1, 5F6, 13G12 and 9B3 with corresponding light chain variable sequences SEQ ID NOs: 5, 13, 21, 29, 37, 45, 53, 61, 69, 77, 85, 93, 101 and 109; and corresponding heavy chain variable sequences - SEQ ID NOS: ID NO: SEQ ID NO: 1, 9, 17, 25, 33, 41, 49, 57, 65, 73, 81, 89, 97 and 105.
  • These antibodies exhibit certain desired functions, such as stimulation of GITR and increased T cell activation and
  • the humanization design of GITR antibody adopts the method of grafting the complementarity determining region.
  • the IMGT database was searched for the human germline antibody sequences with the highest homology to the light and heavy chain variable regions of murine 10E11, 4E4, 4B11 and 3F5 antibodies, respectively.
  • the germline selected for the humanization of the variable region of the light chain of the 10E11 antibody was IGKV3-15*01, and the variable region of the heavy chain was selected as IGHV5-10-1*01.
  • the variable region of the light chain of the 4E4 antibody was selected for humanization.
  • the germline was IGKV2-28*01, and IGHV1-2*06 was chosen for the humanization of the heavy chain variable region.
  • IGKV3-11*01 was chosen for the light chain humanization of the 4B11 antibody, and IGHV1-3*01 was chosen for the heavy chain humanization.
  • 3F5 antibody light chain humanization selects IGKV2-24*01, heavy chain humanization selects IGHV1-2*02.
  • the CDR regions of the murine antibody were retained, and the framework sequence of the murine antibody was replaced with the framework sequence of the human germline antibody.
  • establish the structural model of the mouse antibody and compare each different amino acid position in the human antibody and the corresponding mouse antibody structural model one by one. If the structure is destroyed or changed, the human amino acid sequence is used in this site, otherwise the corresponding murine sequence is used in this site (ie, backmutation to murine sequence).
  • Part of the amino acids in the framework regions of the 10E11, 4E4, 4B11 and 3F5 humanized antibodies were backmutated to murine sequences according to structural modeling.
  • the amino acid sequences of the CDRs of the human antibodies in the above template were substituted with the amino acid sequences of the CDRs of the mouse 4E4, 10E11, 4B11 and 3F5 antibodies.
  • the VH and VL frameworks of the template human antibodies described above were grafted with the essential amino acid sequences of VH and VL of mouse 4E4, 10E11, 4B11 and 3F5 antibodies to obtain functional humanized antibodies.
  • the VH and VL of 4E4, 10E11, 4B11 and 3F5 several sites of the framework amino acids of the template human antibodies described above were backmutated to the corresponding amino acid sequences in mouse 4E4, 10E11, 4B11 and 3F5 antibodies.
  • the 24th Gly of the humanized heavy chain of the 10E11 antibody is backmutated to Ala
  • the 28th Ser is backmutated to Pro
  • the 29th Phe is backmutated to Val
  • the 30th Thr is backmutated to Ser
  • the 48th Met is backmutated is Ile
  • the 69th Ile is backmutated to Phe
  • the 73rd Lys is backmutated to Ile
  • the 93rd Ala is backmutated to Ser.
  • Tyr at position 49 of the humanized light chain of the 10E11 antibody was backmutated to Ser.
  • the 4E4 antibody humanized heavy chain has a back-mutation of Met at position 48 to Ile, back-mutation of Val at position 67 to Ala, back-mutation of Met at position 69 to Leu, back-mutation of Arg at position 71 to Val, and back-mutation of Thr at position 73 for Lys.
  • the Ile at position 2 of the humanized light chain of the 4E4 antibody was backmutated to Val.
  • the 4B11 antibody humanized heavy chain has a reverse mutation of Tyr at position 27 to Phe, Met at position 48 to Ile, Val at position 67 to Ala, Ile at position 69 to Leu, and Arg at position 71. Ser, and Thr at position 73 was backmutated to Lys.
  • Tyr at position 49 of the humanized light chain of the 4B11 antibody was backmutated to Lys.
  • the 48th position of the humanized heavy chain of the 3F5 antibody is backmutated to Ile, the 67th position of Val is backmutated to Ala, the 69th position of Met is backmutated to Leu, the 71st position of Arg is backmutated to Val, and the 73rd position of Thr is backmutated to Val for Lys.
  • Leu at position 46 of the humanized light chain of the 3F5 antibody was backmutated to Arg.
  • the amino acid sequence numbers of the variable regions of the heavy and light chains of the 10E11 humanized antibody are SEQ ID NO: 119 and SEQ ID NO: 129, respectively.
  • the amino acid sequence numbers of the variable regions of the heavy and light chains of the 4E4 humanized antibody are respectively SEQ ID NO: 119 and SEQ ID NO: 129.
  • the amino acid sequence numbers of the variable regions of the heavy and light chains of the 4B11 humanized antibody are SEQ ID NO: 115 and SEQ ID NO: 116, respectively.
  • the heavy chain of the 3F5 humanized antibody The amino acid sequence numbers of the variable region of the light chain and the light chain are SEQ ID NO: 117 and SEQ ID NO: 118, respectively.
  • 10E11, 4E4, 4B11, and 3F5 humanized antibodies were constructed to the IgG1 subtype; humanized 4E4, 10E11, and 4B11 humanized IgG1 (hlgG1) versions, humanized 10E11 hIgG1 (3A) versions, and human hIgG4 versions of the 10E11 and 3F5 antibodies were derived: h10E11-hIgG1, h4E4-hIgG1, h4B11-hIgG1, h10E11-hIgG1 (3A), h10E11-hIgG4 and h3F5-hIgG4.
  • the amino acid sequences include h4E4-IgG1 [SEQ ID NOs: 121 (full length heavy chain) and 122 (full length light chain)], h10E11-IgG1 [(SEQ ID NOs: 127 and 128 (full length heavy chain) and 131 (full length heavy chain)] long light chain)), h10E11-IgG1(3A) [(SEQ ID NOs: 129 (full length heavy chain) and 131 (full length light chain)), h10E11-IgG4 [(SEQ ID NOs: 130 (full length heavy chain) ) and 131 (full-length light chain)), h4B11-IgG1 [(SEQ ID NOs: 123 (full-length heavy chain) and 124 (full-length light chain)) and h3F5-IgG1 [(SEQ ID NOs: 125 (full-length heavy chain) and 126 (full-length light chain)), as shown
  • DNA encoding humanized antibody light and heavy chains was synthesized and cloned into the expression vector pcDNA3.1 (Invitrogen, CAT: #V-790) .
  • Freestyle 293 cells 200 mL, 10 6 /mL were transfected with 100 ⁇ g of each humanized heavy and light chain expression plasmid and cultured for 6 days.
  • the humanized antibody in the supernatant was then purified using a Protein-G column (GE Healthcare).
  • Binding of humanized antibodies to GITR was determined using ELISA and FACS, GITR ligand-blocked activity was determined using FACS, and immune activation responses of humanized antibodies were analyzed based on the Jurkat-GITR-NF- ⁇ B cell reporter gene assay.
  • binding and specificity of the antibodies were determined by ELISA and FACS-based binding assays and GITR-L blocking assays in Jurkat-GITR cells. The methods used were similar to those described in Examples 2 and 4 above.
  • the humanized antibody showed significant binding to GITR, as shown in Figure 1, the EC50 of humanized anti-GITR antibody binding to GITR-mFc protein was detected by ELISA; humanized
  • the anti-GITR antibodies include h10E11(AV)-hIgG1(3A), h10E11(AV)-hIgG4, h4E4-hIgG1, h4B11-hIgG1 and h3F5-hIgG1.
  • the upper part of the graph shows absorbance values over a range of concentrations of humanized antibody or control hIgG1.
  • the tested concentrations of each antibody were 20000ng/mL, 2000ng/mL, 200ng/mL, 20ng/mL, 2ng/mL, 0.2ng/mL, 0.02ng/mL, 0.002ng/mL, 0.0002 ng/mL and 0.00002ng/mL.
  • the table at the bottom of the figure shows the calculated EC50 for each antibody tested.
  • all the humanized antibodies showed significant binding to plate-coated GITR-mFc, with stronger binding for h4B11-hlgG1 and h10E11(AV)-hIgG1 (3A).
  • Figure 2 shows FACS-based binding to GITR on Jurkat-GITR cells.
  • the bottom panel provides the EC50 for each humanized antibody.
  • 4B11-hlgGl and 10E11(AV)-hlgGl were the major binders based on FACS analysis.
  • Humanized GITR antibodies include h10E11(AV)-hIgG1, h10E11(AV)-hIgG1(3A), h10E11(AV)-hIgG4, h4E4-hIgG1, h4B11-hIgG1, h3F5-hIgG1 and a negative control (human IgG1).
  • the upper part of the graph shows the mean fluorescence index values over a range of humanized antibody or control hIgGl concentrations.
  • the bottom shows the calculated EC50 for each antibody tested.
  • the results showed that all the humanized antibodies showed significant binding to Jurkat-GITR cells.
  • humanized h4E4 and h10E11 (AV) antibodies were the main binders.
  • the tested concentrations of each antibody were 20000ng/mL, 2000ng/mL, 200ng/mL, 20ng/mL, 2ng/mL, 0.2ng/mL, 0.02ng/mL and 0.002ng/mL, respectively.
  • Figure 3 shows the binding of GITR-L to GITR by humanized antibodies on Jurkat-GITR cells.
  • GITR-L concentration used was 0.25ug/ml);
  • GITR antibodies tested included h10E11(AV)-hIgG1, h10E11(AV)-hIgG1(3A), h10E11(AV)-hIgG4, h4E4-hIgG1, h4B11-hIgG1, h3F5-hlgG1 and negative control (human IgG1).
  • the tested concentrations of each antibody were 20000ng/mL, 4000ng/mL, 800ng/mL, 160ng/mL, 32ng/mL, 6.4ng/mL, 1.28ng/mL and 0.256ng/mL, respectively.
  • the results showed that all the humanized GITR antibodies showed a certain blocking activity on the binding of GITR-L to Jurkat-GITR cells.
  • Humanized h10E11(AV) and h3F5 antibodies have better blocking effect.
  • Figure 4 shows the GITR activating activity of humanized anti-GITR antibodies based on the 293T-GITR-NFkb-luc cell reporter gene assay.
  • Anti-GITR antibodies tested included h10E11(AV)-hIgG1(3A), h10E11(AV)-hIgG1, h4E4-hIgG1, h4B11-hIgG1, h3F5-hIgG1 and a negative control (human IgG1).
  • the tested concentrations of each antibody were 20000ng/mL, 4000ng/mL, 800ng/mL, 160ng/mL, 32ng/mL, 6.4ng/mL, 1.28ng/mL and 0.256ng/mL, respectively.
  • the results showed that all the humanized antibodies had strong activation activity on the expression of luciferase reporter gene in 293T-GITR-NFkb-luc cells. Activity was similar between the tested humanized antibodies.
  • the antibodies were first evaluated by their stimulating effect on the GITR signaling pathway in Jurkat-GITR cells.
  • Jurkat-GITR, 293T-OKT3, 293T-CD32A and 293T cells were individually cultured in RPMI 1640 supplemented with 10% FBS.
  • 50 ⁇ l Jurkat-GITR (1x105 cells per well) and 50 ⁇ l 293T-OKT3 (2x104 cells per well) and 50 ⁇ l 293T-CD32A or 293T (2x104 cells per well) cells were added to a single well of a 96-well plate. Presence of diluted GITR antibody. After 48 or 72 hours of incubation at 37°C, the 96-well plate was centrifuged and the supernatant was collected for IL-2 measurement (R&D Systems, cat. no. DY202).
  • Figure 5 shows the results of IL2 release without cross-linking due to activation of Jurkat-GITR cells by humanized anti-GITR antibody.
  • Jurkat-GITR cells were stimulated to some extent by OKT3 in 293T-OKT3 cells.
  • Anti-GITR antibodies tested included h10E11(AV)-hIgG1(3A), h10E11(AV)-hIgG1, h4E4-hIgG1, h4B11-hIgG1, h3F5-hIgG1 and a negative control (human IgG1).
  • the tested concentrations of each antibody were 20000ng/mL, 4000ng/mL, 800ng/mL, 160ng/mL, 32ng/mL, 6.4ng/mL, 1.28ng/mL and 0.256ng/mL, respectively.
  • all humanized antibodies can stimulate the release of IL2 from Jurkat-GITR cells to some extent without cross-linking.
  • Figure 6 shows the results of IL2 release due to activation of Jurkat-GITR cells by humanized anti-GITR antibodies in the presence of cross-linking.
  • the cross-linking factor is CD32a expressed in 293T-CD32a cells.
  • Jurkat-GITR cells were stimulated to some extent by OKT3 in 293T-OKT3 cells.
  • Anti-GITR antibodies tested included h10E11(AV)-hIgG1(3A), h10E11(AV)-hIgG1, h4E4-hIgG1, h4B11-hIgG1, h3F5-hIgG1 and a negative control (human IgG1).
  • the tested concentrations of each antibody were 20000ng/mL, 4000ng/mL, 800ng/mL, 160ng/mL, 32ng/mL, 6.4ng/mL, 1.28ng/mL and 0.256ng/mL, respectively.
  • all the humanized antibodies showed stronger stimulatory activity on IL2 release from Jurkat-GITR cells, especially h4E4-hlgG1.
  • GITR hybridoma antibodies were assessed by their effect on stimulating the GITR signaling pathway in lymphocyte effector cells.
  • Human CD4+ T cells were purified from PBMCs using a CD4+ negative selection kit in RPMI 1640 supplemented with 10% FBS.
  • 96-well plates were coated with goat anti-human IgG (Jackson, catalog number: 109-005-008) and goat anti-mouse IgG (Jackson, catalog number: 115-006-071 ) overnight at room temperature. temperature.
  • Wells were washed and blocked by adding 200 ⁇ L of blocking buffer and incubated for 1 hour at 37°C.
  • Figure 7 shows the effect of humanized anti-GITR antibodies on IL2 production by CD4+ T cells.
  • Anti-GITR antibodies tested included h10E11(AV)-hIgG1(3A), h10E11(AV)-hIgG1, h4E4-hIgG1, h4B11-hIgG1, h3F5-hIgG1 and a negative control (human IgG1).
  • the tested concentrations of each antibody were 20 ng/mL, 2 ng/mL, 0.2 ng/mL and 0.02 ng/mL, respectively.
  • the humanized 10E11, 4B11 and 4E4 antibodies had a significant stimulating effect on the production of the cytokine IL2 by primary CD4 T cells; whereas the humanized 3F5 antibody showed activity in this assay relatively low.
  • Figure 8 shows the effect of humanized anti-GITR antibodies on IFN- ⁇ production by CD4+ T cells.
  • Anti-GITR antibodies tested included h10E11(AV)-hIgG1(3A), h10E11(AV)-hIgG1, h4E4-hIgG1, h4B11-hIgG1, h3F5-hIgG1 and a negative control (human IgG1).
  • the tested concentrations of each antibody were 20 ng/mL, 2 ng/mL, 0.2 ng/mL and 0.02 ng/mL, respectively.
  • the humanized 10E11, 4B11 and 4E4 antibodies had a significant stimulating effect on the production of the cytokine IFN- ⁇ by primary CD4 T cells; while the humanized 3F5 antibody showed in this analysis lower activity.
  • the humanized 3F5 antibody showed relatively low activation activity.
  • Example 12 Biacore kinetic analysis of humanized 4E4, 10E11, 4B11 and 3F5 anti-GITR antibodies
  • GITR and anti-GITR antibodies were measured by Biacore3000 and recorded at a data acquisition rate of 1 Hz.
  • Polyclonal rabbit anti-mouse IgG (GE, BR-1008-38) was diluted with 10 mM sodium acetate pH 5.0 and immobilized on the reference and experimental flow cells of a CM5 biosensor chip using an amine coupling kit (GE, BR10050) to about 15000RU).
  • diluted test antibody 1.5 ⁇ g/mL was injected into the experimental flow cell for 1 min for capture.
  • the GITR analyte series was prepared by diluting the stock solution to 100 nM with flow buffer, followed by a 2-fold serial dilution to 0.78 nM in the same buffer.
  • the analytes were continuously injected into the reference and experimental flow cells for 3 minutes at a flow rate of 30 ⁇ L/min.
  • running buffer 0.05% P20 in PBS
  • the biosensor surface was regenerated by injecting 10 mM pH 1.7 glycine-HCl buffer for 3 min at a flow rate of 10 ⁇ L/min.
  • association and dissociation rate constants (ka and kd) can be determined simultaneously by fitting dual-reference sensorgrams of the entire titration series to the Langmuir model (1:1) using Biaevaluation 4.0 software.
  • Table 10 the humanized anti-GITR antibodies bound human GITR with high affinity. Especially the humanized 4B11 and 10E11.
  • the monoclonal antibodies or antigen-binding fragments thereof of the present invention are used in the preparation of drugs for activating GITR signaling, Drugs that activate T lymphocytes, drugs that increase the expression of IL-2 and INF- ⁇ in T lymphocytes, drugs that block the combination of GITR-L and GITR, and drugs that prevent and treat or adjuvant tumors have good application prospects .

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Abstract

提供一组抗GITR的单克隆抗体及其人源化抗体，可制备用于激活免疫反应药物，尤其是治疗癌症的相关药物。

Description

一组GITR单克隆抗体及其医药用途 技术领域
本发明属于肿瘤及免疫学药物领域，具体地，本发明涉及一组抗GITR的单克隆抗体及其医药用途。
技术背景
GITR是糖皮质激素诱导的TNFR相关蛋白，也称为TNFRSF18或CD357；它是一种I型跨膜蛋白，在Treg细胞上有高水平表达，而在初始和记忆T细胞上有低水平的表达[Shimizu等，(2002)，Nat.Immunol.3：135-42；Nocentini等，(2009)，Adv.Exp.Med.Biol.647：156-73；McHughet等，(2002)，Immunity 16：311-23；Nocentini等，(2005)，Eur J Immunol 35：1016–1022]。激活后，初始T细胞和TregT细胞迅速上调GITR的表达(Shimizu等，(2002),Nat.Immunol.3：135-42；Krausz等，(2007,Scientific World Journal 7：533-66)。在活化的先天性免疫细胞(例如天然杀伤细胞，巨噬细胞和树突细胞(DC)上也有从低到中等水平的GITR表达[Clouthier等，(2014),Growth Factor Rev.25：91-106]。GITR的配体(GITRL)是属于TNF超家族的II型跨膜蛋白，它在包括B细胞，巨噬细胞和树突状细胞(DC)在内的内皮细胞以及活化的抗原呈递细胞(APC)的细胞表面上观察到一定量的表达[Yu等，(2003),Biochem.Biophys.Res.Commun.310：433-438；Toneet等，(2003),Proc.Natl.Acad.Sci.USA 100：15059-15064]。
通过GITRL或GITR激动剂抗体与GITR的相互作用，GITR可被刺激从而增强TCR诱导的T细胞增殖和细胞因子产生的作用[Ronchetti等，(2004)，Eur.J.Immunol.34：613-622；Tone等,(2003)，Proc.Natl.Acad.Sci.USA 100：15059-15064；Stephens等，(2004)，J.Immunol.173：5008-5020]，以及降低效应T细胞对Treg细胞介导的免疫抑制作用的敏感性[Ronchetti等，(2007)，Eur.J.Immunol.37：1165-1169；Stephens等，(2004),J.Immunol.173：5008-5020]。GITR的激活可募集TNFR相关联的因子2(TRAF 2)和5(TRAF5)，并诱导激活转录因子NF-κB和MAP激酶诱导的信号传导通路[Snellet等，(2011)，Immunol.Rev.244：197-217；Snell等，(2010),J.Immunol.185：7223-34]，以及诱导抗凋亡的Bcl-XL蛋白的转录，从而减少由细胞激活作用所导致的T细胞的死亡[Ronchetti等人，(2004)，Eur.J.Immunol.34：613-622；Esparza等，(2006)，J.Biol.Chem.281：8555-64]。GITR的激活还可进一步导致依赖于NFkB的 TRAF6活化以及IL-9的产生，从而增强DC细胞的功能以及激活T淋巴细胞的细胞毒性反应[Kim等，(2015)，Nat.Med.21：1010-7]。GITR信号传导也可促进FoxP3的降解[Cohenet等，(2010),PLoS ONE 5：10436；Schaer等，(2013),Cancer Immunology Research 1：320-331]。以及c-Jun N末端激酶(JNK)的磷酸化[Joetham等，(2012)，J Biol.Chem.287：17100-17108]；从而，进一步抑制Treg细胞对免疫的抑制功能。另外，也有报道显示抗GITR抗体可逆转Treg细胞对B细胞介导的抗体产生作用的抑制效应[Cava等，(2004)，J.Immunol.173：3542-3548]。
临床前研究表明，GITR信号传导通路的激活可以显著增强肿瘤的消除作用。其效果取决于免疫效应细胞的激活[Ramirez等，(2006),J Immunol 176：6434-6442；Boczkowski等，(2009),Cancer Gene Ther.16：900-911；Mitsui等，(2010)，Clin.Cancer Res.16：2781-91],也取决于对Treg细胞免疫抑制活性的去除或压制[Cohen等，(2010)，PLoS ONE 5:e10436；Coe等，(2010),Cancer Immunol.Immunother.59：1367-77；Xiao等，(2015),Nat.Commun.6：8266]。GITR激活型抗体被单独使用时，可减少Treg细胞进入肿瘤组织,并使肿瘤内Treg细胞失去foxp3的表达；从而局部性地消除肿瘤内Treg细胞的免疫抑制作用。最终，在肿瘤微环境中，Teff/Treg细胞比率得到提高，并增强了Teff细胞的活性和功能(Cohen等，(2010)，PLoS ONE 5：e10436)。在卵巢癌模型中，抗GITR和抗PD-1抗体的联合疗法可整体提高病人的存活率；在治疗90天后，无肿瘤的小鼠达到20％。分析小鼠肿瘤内进入的白细胞(TILs)的结果，发现CD4+和CD8+效应记忆T细胞的数目得到显着的增加，同时，Treg免疫调控细胞的数目显著的减少[Lu等，(2014)，J.Transl.Med.12：36-47]。虽然单药治疗的效果对于该GITR抗体来说不是很显著，它和抗CTLA4抗体的联合用药在MethA和CT26的肿瘤模型中出现了显著增强的协同抗肿瘤作用[Ko等，(2005),J.Exp.Med.202：885-91；Mitsui等，(2010),Clin.Cancer Res.16：2781-91]。
发明内容
在此基础上，本发明通过杂交瘤技术获得了一组具有激活GITR和免疫细胞活性的，而且可以阻断GITR－L与GITR相互作用的效果优异的抗GITR单克隆抗体；并成功的对相关抗体进行了人源化改造。所述抗体在制备用于刺激和 增强免疫反应的药物以及治疗癌症相关药物方面有显著的应用前景。
本发明提供了一组抗GITR单克隆抗体或其抗原结合片段，包括重链和轻链，其特征在于，所述重链的CDR1的氨基酸序列选自SEQ ID NO：2,10,18,26,34,42,50,58,66,74,82,90,98和106中的一种；所述重链的CDR2的氨基酸序列选自SEQ ID NO：3,11,19,27,35,43,51,59,67,75,83,91,99和107中的一种；所述重链的CDR3的氨基酸序列选自SEQ ID NO：4,12,20,28,36,44,52,60,68,76,84,92,100和108中的一种；所述轻链的CDR1氨基酸序列选自SEQ ID NO：6,14,22,30,38,46,54,62,70,78,86,94,102和110中的一种；所述轻链的CDR2的氨基酸序列选自SEQ ID NO：7,15,23,31,39,47,55,63,71,79,87,95,103和111中的一种；所述轻链的CDR3的氨基酸序列选自SEQ ID NO：8,16,24,32,40,48,56,64,72,80,88,96,104和112中的一种；其中所述抗原结合片段的重链和轻链包含分别跨越所述抗体的重链和轻链的CDR1到CDR3的氨基酸序列。
进一步的，本发明公开了上述的一组GITR单克隆抗体或其抗原结合片段，其特征在于，所述重链可变区的氨基酸序列选自SEQ ID NO：1,9,17,25,33,41,49,57,65,73,81,89,97和105中的一种；所述轻链可变区的氨基酸序列选自SEQ ID NO：5,13,21,29,37,45,53,61,69,77,85,93,101和109中的一种。
进一步的，本发明公开了对上述抗GITR单克隆抗体或其抗原结合片段的重链和轻链进行人源化改造，所述经过人源化改造的重链可变区的氨基酸序列选自SEQ ID NO：113,115,117和119中的一种；所述经过人源化改造的轻链可变区的氨基酸序列选自SEQ ID NO：114,116,118和120中的一种。
进一步的，本发明公开了上述经过人源化改造的一组抗GITR单克隆抗体或其抗原结合片段，所述人源化改造的重链的全长氨基酸选自SEQ ID NO：121,123，125，127，128，129和130中的一种；所述人源化改造的轻链的全长氨基酸选自SEQ ID NO：122,124，126和131中的一种。
进一步的，本发明相应公开了编码上述GITR单克隆抗体或其抗原结合片段的的核苷酸序列。
进一步的，本发明公开了上述抗GITR片段在制备如下药物中的用途：激活T淋巴细胞的药物、阻断GITR-L与GITR结合的药物、或者提高T淋巴细胞中IL-2和IFN-γ表达的药物。
进一步的，本发明公开了一种单克隆抗体偶联物，包括单克隆抗体和偶联部分，其中，所述单克隆抗体为权利要求1-4任一项所述的GITR单克隆抗体或其抗原结合片段，所述偶联部分为选自放射性核素、药物、毒素、细胞因子、细胞 因子受体片段、酶、荧光素、和生物素中的一种或多种。
进一步的，本发明公开了上述单克隆抗体偶联物在制备如下药物中的用途：激活T淋巴细胞的药物、阻断GITR-L与GITR结合的药物、或者提高T淋巴细胞中IL-2和IFN-γ表达的药物。
进一步的，本发明公开了上述单克隆抗体偶联物在制备预防和/或治疗和/或辅助治疗肿瘤的药物中的用途。
本发明的进步性表现为：本发明采用哺乳动物细胞表达系统制备重组GITR作为抗原免疫小鼠，将小鼠脾脏细胞与骨髓瘤细胞融合获得杂交瘤细胞。通过对大量杂交瘤细胞的多次克隆及筛选后，得到一些单克隆杂交瘤细胞株。这些杂交瘤细胞株能够产生和GITR特异性高亲和力结合的单克隆抗体，能促进混合淋巴细胞反应中IL-2，INF-γ的分泌，以及能够有效阻断GITR－L与GITR的结合，并且通过RT-PCR克隆编码抗体轻链和重链可变区的基因，采用互补决定簇嫁接方法构建人源化抗体。体外功能试验表明，这些人源化的GITR抗体能特异性高亲和力结合GITR蛋白，能显著促进T细胞分泌细胞因子IL-2和INF-γ，并且能阻断GITR－L与GITR的结合。以上实验结果表明本发明所述的单克隆抗体或其抗原结合片段，或者包含本发明所述单克隆抗体或其抗原结合片段的单克隆抗体偶联物在制备激活T淋巴细胞的药物、提高T淋巴细胞表达IL-2和INF-γ的药物、阻断GITR－L与GITR结合的药物、以及在预防和治疗或者辅助治疗肿瘤的药物方面具有良好的应用前景。
附图说明
图1为用ELISA方法检测人源化抗GITR抗体与GITR-mFc蛋白结合的EC50；
图2显示了人源化GITR抗体与细胞膜上GITR相结合的FACS测试结果；
图3显示了人源化GITR抗体对GITR-L与Jurkat-GITR细胞的结合的阻断作用；
图4显示了基于293T-GITR-NFkb-luc细胞报告基因测定法的人源化抗GITR抗体的GITR激活活性；
图5是显示了人源化GITR抗体在没有交联的情况下活化Jurkat-GITR细胞使其释放细胞因子IL2的结果；
图6表明了人源化GITR抗体在有交联的情况下活化Jurkat-GITR细胞使其释放细胞因子IL2的结果；
图7显示了人源化GITR抗体对CD4T细胞产生细胞因子IL2的影响；
图8展现的是人源化GITR抗体对CD4T细胞产生细胞因子IFN-γ的影响。
具体实施方式
下面通过具体实施方式来进一步说明本发明的技术方案。本领域技术人员应该明了，所述实施例仅仅是帮助理解本发明，不应视为对本发明的具体限 制；下述实施例中所使用的实验方法如无特殊说明，均为常规方法；下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到；本发明所涉及的抗体的氨基酸见表1-3。
表1 GITR抗体轻链CDR序列
Figure PCTCN2021080981-appb-000001
Figure PCTCN2021080981-appb-000002
表2 GITR抗体重链CDR序列：
Figure PCTCN2021080981-appb-000003
Figure PCTCN2021080981-appb-000004
Figure PCTCN2021080981-appb-000005
表3 抗体的轻链和重链可变区序列以及完整抗体序列
Figure PCTCN2021080981-appb-000006
Figure PCTCN2021080981-appb-000007
Figure PCTCN2021080981-appb-000008
Figure PCTCN2021080981-appb-000009
Figure PCTCN2021080981-appb-000010
Figure PCTCN2021080981-appb-000011
实施例1
抗GITR杂交瘤抗体的产生。
用东大生物表达纯化的人源GITR(ECD)-mFc(Kim et al.,2007,Oncol Rep.18:1189).融合蛋白作为抗原，与等体积完全弗氏佐剂(Sigma,Cat No:F5581)充分乳化后，经皮下免疫6-8周龄Balb/c小鼠(购自昭衍(苏州)新药研究中心有限公司)，抗原免疫量为50μg/只；随后每隔2周，用相同剂量的抗原与不完全弗氏佐剂(Sigma,Cat No:F5506)充分乳化后，经皮下免疫小鼠三次。免疫三次后测定小鼠血清效价，融合前3天经腹腔进行加强免疫。以PEG Hybri-Max(Sigma,Cat No:7181)作为融合剂，将小鼠脾脏细胞与SP2/0细胞按照4:1的比例混合。将融合后的细胞加入到96孔板中(1×10 5细胞/孔)，每孔含有0.1mL 1X HAT培养基(Invitrogen,Cat No:21060-017)。在第3天加入0.1mL HT(Invitrogen,Cat No:11067-030)培养基，在第7天吸掉96孔板中的培养基，补加0.2mL新鲜的HT培养基。在第9天，收取上清液，用于下一步的ELISA和FACS检测。
实施例2
基于ELISA、FACS和报告基因测试的多克隆杂交瘤筛选分析。
这些测试的目的是筛选出能与GITR结合并能阻断GITR－L与GITR的相互作用以及能显著激活GITR分子活性的多克隆杂交瘤，选择其中具高结合力和高刺激活性的多克隆杂交瘤进行亚克隆，从而得到所需的单克隆杂交瘤抗体。
在将具高ELISA滴度的免疫小鼠脾细胞与SP2/0细胞融合以及培养7天后，对杂交瘤细胞首先进行ELISA和FACS测定以发现和筛选能与GITR结合的阳性杂交瘤，然后初步使用293T荧光素酶报告基因测试筛选具有功能性应答反应的多克隆杂交瘤。此项工作总共鉴定出了四十几个与GITR结合较强的多克隆杂交瘤抗体，经报告基因测试后，选择其中结合力和刺激活性显著的多克隆杂交瘤进 行下一步的亚克隆筛选。与GITR结合较强的杂交瘤细胞被冷冻保存在液氮中。
关于ELISA检测：用GITR-ECD-hFc包被96孔ELISA板(Corning，Cat No:9018)，置4度过夜，用洗涤缓冲液(PBS+0.05％Tween20)洗涤3次后，加入封闭缓冲液(PBS+1％BSA(Sigma,Cat No:V90093)孵育1小时；洗涤96孔板3次；加入杂交瘤上清孵育1小时，洗涤3次；每孔加入100μL 1:10000倍稀释的羊抗鼠IgG二抗(Thermo,Cat No:31432)，室温孵育1小时后洗涤3次；每孔加入100μL TMB(北京百奥赛博，Cat No:ES-002)显色3分钟，再加入100μL/孔的终止液(2N H 2SO 4)终止反应，用Tecan Spark酶标仪测定各样品的OD450×信号。
关于抗体结合力的FACS分析：取50μL在上述ELISA检测中呈阳性的杂交瘤上清与50μL 293T-GITR细胞混合(1×10 5细胞/孔)加入到U形底96孔板中孵育1小时，用FACS缓冲液(PBS+3％FCS)离心洗涤2次后加入1:400倍稀释的PE标记羊抗鼠二抗(Biolegend，Cat No:405307)，孵育30分钟，经FACS缓冲液洗涤后，用BD C6流式细胞仪检测293T-GITR细胞的PE信号。
关于抗体阻断作用的FACS分析：使用BD C6进行FACS分析以确定杂交瘤抗体对经过生物素标记的人源GITR配体结合到Jurkat-hGITR细胞上的阻断作用。将Jurkat-hGITR细胞悬浮在FACS缓冲液(含3％FBS的PBS)中。将稀释的杂交瘤抗体添加到细胞悬液中，并在4℃下于96孔板中孵育1小时。将生物素标记的人GITR配体蛋白添加到96孔板的孔中，并在4℃下孵育1小时。将孵育后的96孔板洗涤3次，并加入小鼠抗生物素PE抗体(Biolgend，目录号409004)，并在4℃下孵育0.5小时。将96孔板中的细胞洗涤3次，最后用BD C6进行分析。
关于基于Jurkat-GITR-NF-κB细胞的报告基因测试加以确认杂交瘤抗GITR抗体在免疫激活方面的作用的分析：通过对抗GITR杂交瘤上清液，用Jurkat-GITR-NF-κB细胞系进行报告基因的FACS分析。每个反应使用25000Jurkat-GITR-NF-κB细胞，将这些细胞加入到96孔培养板的每个孔中，然后在每个孔中再加入50μL杂交瘤上清液，并在37℃下孵育4小时。用50μLBright Glo试剂(Promega，目录号：E2620)裂解细胞，并使用Tecan Spark在发光条件下读取信号。
实施例3
多克隆杂交瘤细胞的亚克隆，GITR单克隆杂交瘤细胞的扩增培养与抗体纯化以及单克隆杂交瘤抗体的DNA序列分析。
亚克隆的目的是为了建立单克隆杂交瘤细胞以获得单克隆抗体克隆。我们的亚克隆方法是基于有限稀释的程序，在96孔板中获得产生单个孔中张单个克隆的杂交瘤细胞。进行多轮(3轮)的亚克隆后，确保所得到的单克隆没有其它的细胞成分。对于每一轮亚克隆，均通过ELISA、FACS以及报告基因激活活性的检测。三轮亚克隆后，对于保持较高GITR结合力宇刺激活性的单克隆细胞，部分用于冻存，另一部分用于细胞扩增培养以获取足够量的单克隆抗体进行纯化以及进一步的测试。
对总共14种抗GITR单克隆杂交瘤细胞进行了培养及抗体纯化，包括4E1、8H4、4E4、3F5、4C1、7D7、7E11、10E11、3G12、4B11、5F1、5F6，13G12和9B3。杂交瘤细胞是在含有10％胎牛血清，1％青霉素/链霉素，2％L-谷氨酰胺和1％调节NaHCO 3溶液的Dulbecco's Modified Eagle's培养基(GIBCO；Invitrogen Corporation，Carlsbad，CA)中培养。然后将所选择的杂交瘤细胞适应于无血清培养基中，并使用Protein-G柱(GE Healthcare)从上清液中纯化抗体。用PBS洗涤后，用0.1M甘氨酸pH3.0洗脱结合的抗体，然后用2.0M Tris中和pH。Ultra-15离心浓缩器(Amicon)用于缓冲液交换和抗体浓缩。
实施例4
对纯化的4E1、8H4、4E4、3F5、4C1、7D7、7E11、10E11、3G12、4B11、5F1、5F6、13G12和9B3的杂交瘤抗hGITR单克隆抗体进行了进一步的GITR结合力、GITR配体阻断作用，基于报告基因的GITR激活活性，以及与猴源GITR的交叉结合特性的测试分析，实验涉及ELISA、FACS以及基于使用Jurkat-GITR-NF-κB细胞的报告基因激活分析。
基于ELISA和FACS方法的测试分析；所用方法与上述实施例2中所述的方法相似，不同的是这里的测试使用了测定浓度的纯化抗体，运用系列稀释的抗体浓度与相应的结果，可以计算出EC 50和IC50值。表4-7显示了14种抗体的测试结果。另外，对与猴源GITR交叉结合特性的分析表明，所选的14种抗体与猴源GITR有交叉结合反应。
表4.杂交瘤抗GITR抗体基于ELISA结合力的EC50
ng/ml 4E1 8H4 4E4 3F5 4C1 7D7 7E11 10E11 3G12 4B11 5F1 5F6 13G12 9B3
EC50 0.067 1.338 1.705 2.314 1.932 1.037 1.144 4.733 1.628 0.883 2.039 1.259 0.277 0.523
表5.杂交瘤抗GITR抗体基于FACS结合力的EC50
ng/ml 4E1 8H4 4E4 3F5 4C1 7D7 7E11 10E11 3G12 4B11 5F1 5F6 13G12 9B3
EC50 0.416 20.41 19.92 6.026 8.3 6.387 7.844 9.173 33.06 15.67 51.74 29.35 77.31 1.923
表6.杂交瘤抗GITR抗体基于FACS分析对GITR-L与GITR结合的阻断作用IC50
ng/ml 4E1 8H4 4E4 3F5 4C1 7D7 7E11 10E11 3G12 4B11 5F1 5F6 13G12 9B3
EC50 12.91 457.6 112.7 906.2 97.29 102.3 70.82 89.77 322.0 318.5 1.513 118.2 4002 41.35
表7.杂交瘤抗GITR抗体在Jurkat-GITR-NF-κB细胞报告基因测试分析中的刺激活性
Activity 4E1 8H4 4E4 3F5 4C1 7D7 7E11 10E11 3G12 4B11 5F1 5F6 13G12 9B3
Luc_signal ++++ ++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ ++ +++
表中不同的+数代表活性的强弱(更多的+表示更强的活性)
实施例5
杂交瘤GITR抗体在激活Jurkat细胞IL-2细胞因子释放方面的活性测试。
为了确定杂交瘤抗GITR抗体的免疫激活活性，Jurkat-GITR细胞的IL2细胞因子释放被用作GITR信号传导通路活化的的评价指标之一。在补充有10％FBS的RPMI 1640中进行Jurkat-GITR细胞培养。在培养前一天，将96孔板在室温下用山羊抗人IgG(Jackson，目录号：109-005-008)和山羊抗小鼠IgG(Jackson，目录号：115-006-071)在常温下包被过夜。洗涤板孔后，添加200μL封闭缓冲液，在37℃下封闭孵育1小时。将板用PBS洗涤3次后，在37℃下将40ng/mL OKT3(ebioscience，目录号：16-0037-85)加入孔中孵育1小时。再用PBS洗涤板后，在有或没有稀释GITR抗体的情况下，将100μl Jurkat-GITR细胞添加到96孔板上的每个孔中(每孔1x105个细胞)。在37℃下孵育48或72小时后，将96孔板离心并收集上清液以测量IL-2(R&D Systems，目录号DY202)。结果示于表8。
表8.基于Jurkat-GITR细胞IL-2释放测定的杂交瘤抗GITR抗体对免疫细胞的激活活性
Activity 4E1 8H4 4E4 3F5 4C1 7D7 7E11 10E11 3G12 4B11 5F1 5F6 13G12 9B3
IL2 ++ +++ ++++ +++ ++ +++ ++ +++ ++ +++ ++ ++ + +++
表中不同的+数代表活性的强弱(更多的+表示更强的活性)
实施例6
测试杂交瘤抗GITR抗体对CD4T细胞激活及产生细胞因子的影响。
为了进一步评估杂交瘤GITR抗体在激活GITR信号通路中的活性，本研究用原代淋巴效应细胞测试了抗体对免疫细胞释放IL2以及IFNγ细胞因子的促进作用。使用CD4T细胞的阴性选择提纯试剂盒以及补充有10％FBS的RPMI 1640培养液，在人源PBMC的基础上，进一步纯化得到人的CD4T细胞。在提纯T细胞的前一天，将96孔板在室温下用山羊抗人IgG(Jackson，目录号：109-005-008)和山羊抗小鼠IgG(Jackson，目录号：115-006-071)在常温下包被过夜。洗涤板孔后，添加200μL封闭缓冲液，在37℃下封闭孵育1小时。将板用PBS洗涤3次后，在37℃下将40ng/mL OKT3(Ebioscience，目录号：16-0037-85)加入孔中孵育1小时。用PBS洗涤板后，在存在或不存在稀释的GITR抗体的情况下，将100μl CD4T细胞添加到96孔板上的每个孔中(每孔1x105个细胞)。在37℃下孵育48或72小时后，将96孔板离心，并收集上清液，然后使用ELISA试剂盒(R&D Systems，Cat。Cat)测量IL-2(R&D Systems，目录号：DY202)和IFN-γ的产生编号：DY285)。结果显示于表9。
表9：基于检测原代CD4T细胞IL-2和IFNg释放的杂交瘤抗GITR抗体对免疫细胞的激活活性。
Activity 4E1 8H4 4E4 3F5 4C1 7D7 7E11 10E11 3G12 4B11 5F1 5F6 13G12 9B3
IL2 +++ NA +++ NA ++ ++++ ++ +++ ++ ++++ ++ ++ NA ++++
IFN-g +++ NA ++++ NA +++/- +++++ +++ +++ +++ ++++ +++ ++++/- NA +++
表中不同的+数代表活性的强弱(更多的+表示更强的活性)
实施例7
GITR杂交瘤单克隆抗体cDNA克隆及序列分析。
对14种抗GITR单克隆杂交瘤抗体，4E1、8H4、4E4、3F5、4C1、7D7、7E11、10E11、3G12、4B11、5F1、5F6，13G12和9B3，进行了DNA序列检测。其相应的氨基酸序列分析结果被列于表1、表2和表3中。
GITR抗体可变区基因的克隆分析包括以下步骤：用TRIzon(Cwbiotech,Cat No:CW0580)裂解GITR单克隆杂交瘤细胞株，提取杂交瘤细胞的总RNA。用HiFi Script cDNA合成试剂盒(Cwbiotech,Cat No:CW2569)将杂交瘤细胞的RNA反转录为cDNA。以cDNA为模板，用简并引物通过PCR方法(Kettleborough et al.(1993)Eur.J Immunology 23:206-211；Strebe et al.(2010)Antibody Engineering 1:3-14)扩增抗体的重链和轻链的可变区基因。反应在S1000TM热循环仪(Bio-Rad，CAT#：184-2000)中以30个循环进行：94℃，1.5分钟变性；50℃，退火1分钟；在72℃和1分钟下进行合成。在第30个循环结束时，将反应混合物在72℃下再温育7分钟加以延伸。
将PCR混合物在含有0.5μg/ml溴化乙锭的1％琼脂糖/Tris-硼酸盐凝胶中进行电泳。从凝胶上切出具有预期大小的DNA片段(重链和轻链约400bp)并纯化。将3μl纯化的PCR产物克隆到pMD-18T载体(Takara，CAT#：D101A)中，转化DH5α感受态细胞，涂板并置37℃过夜培养。从培养板上挑取单克隆，扩大培养后抽提质粒，测定抗体的基因序列。根据抗体的基因序列，分析其互补决定簇(CDR)和骨架区。
抗体的重链和轻链的CDR区氨基酸序列见表1和表2；抗体的重链和轻链的可变区全长氨基酸序列见表3。从相应的杂交瘤克隆中扩增的是14种抗体的可变区序列(见表)：4E1、8H4、4E4、3F5、4C1、7D7、7E11、10E11、3G12、4B11、5F1、5F6、13G12和9B3，具有相应的轻链可变序列SEQ ID NO：5,13,21,29,37,45,53,61,69,77,85,93,101和109；以及相应的重链可变序列-SEQ ID NO：SEQ ID NO：1,9,17,25,33,41,49,57,65,73,81,89,97和105。这些抗体表现出某些所需的功能，例如刺激GITR和增加T细胞活化和细胞因子释放。
实施例8
GITR单克隆杂交瘤抗体的人源化。
对14种抗GITR单克隆杂交瘤抗体，4E1、8H4、4E4、3F5、4C1、7D7、7E11、10E11、3G12、4B11、5F1、5F6，13G12和9B3进行综合(包括GITR结合力、GITR配体的阻断作用、对GITR和免疫细胞的激活活性、抗体的DNA序列等)的分析和比较后，我们选择了其中的4个，包括10E11、4E4、4B11和3F5，作了进一步的GITR抗体人源化改造。
GITR抗体的人源化设计采用了互补决定区嫁接法。首先，在IMGT数据库中分别搜寻与鼠源10E11、4E4、4B11和3F5抗体的轻、重链可变区同源性最高 的人胚系抗体(germline antibody)序列。10E11抗体轻链可变区人源化选取的胚系为IGKV3-15*01，重链可变区人源化选取IGHV5-10-1*01。4E4抗体轻链可变区人源化选取的胚系为IGKV2-28*01，重链可变区人源化选取IGHV1-2*06。4B11抗体轻链人源化选取IGKV3-11*01，重链人源化选取IGHV1-3*01。3F5抗体轻链人源化选取IGKV2-24*01，重链人源化选取IGHV1-2*02。保留鼠源抗体的CDR区，将鼠源抗体的框架区(framework)序列用人胚系抗体的框架区序列置换。其次，建立鼠源抗体的结构模型，逐个对比人源抗体与相应鼠源抗体结构模型中的每个不同氨基酸位点，如果在框架区的某个位点采用人的氨基酸序列没有导致CDR区域空间结构的破坏或改变，则该位点使用人的氨基酸序列，否则在该位点使用对应的鼠源序列(即回复突变为鼠源序列)。根据结构模拟，将10E11、4E4、4B11和3F5人源化抗体框架区的部分氨基酸回复突变为鼠源序列。
这样，上述模板中人抗体的CDR氨基酸序列被小鼠4E4、10E11、4B11和3F5抗体的CDR的氨基酸序列取代。另外，将上述模板人抗体VH和VL的框架移植有小鼠4E4、10E11、4B11和3F5抗体的VH和VL的必需氨基酸序列，以得到功能性人源化抗体。另外，关于于4E4、10E11、4B11和3F5的VH和VL，上述模板人抗体的框架氨基酸的几个位点被反突变为小鼠4E4、10E11、4B11和3F5抗体中的相应氨基酸序列。
10E11抗体人源化重链的第24位Gly回复突变为Ala，第28位Ser回复突变为Pro，第29位Phe回复突变为Val，第30位Thr回复突变为Ser，第48位Met回复突变为Ile，第69位Ile回复突变为Phe，第73位Lys回复突变为Ile，第93位Ala回复突变为Ser。10E11抗体人源化轻链的第49位Tyr回复突变为Ser。4E4抗体人源化重链的第48位Met回复突变为Ile，第67位Val回复突变为Ala，第69位Met回复突变为Leu，第71位Arg回复突变为Val，第73位Thr回复突变为Lys。4E4抗体人源化轻链的第2位Ile回复突变为Val。4B11抗体人源化重链的第27位Tyr回复突变为Phe，第48位Met回复突变为Ile，第67位Val回复突变为Ala，第69位Ile回复突变为Leu，第71位Arg回复突变为Ser，第73位Thr回复突变为Lys。4B11抗体人源化轻链的第49位Tyr回复突变为Lys。3F5抗体人源化重链的第48位Met回复突变为Ile，第67位Val回复突变为Ala，第69位Met回复突变为Leu，第71位Arg回复突变为Val，第73位Thr回复突变为Lys。3F5抗体人源化轻链的第46位Leu回复突变为Arg。
10E11人源化抗体重链和轻链的可变区氨基酸序列号分别为SEQ ID NO：119和SEQ ID NO：129。4E4人源化抗体重链和轻链的可变区氨基酸序列号分 别为SEQ ID NO：113和SEQ ID NO：114。4B11人源化抗体重链和轻链的可变区氨基酸序列号分别为SEQ ID NO：115和SEQ ID NO：116。3F5人源化抗体重链和轻链的可变区氨基酸序列号分别为SEQ ID NO：117和SEQ ID NO：118。将10E11、4E4、4B11和3F5人源化抗体构建为IgG1亚型；产生了人源化的4E4、10E11和4B11的人源IgG1(hIgG1)版本，人源化10E11的hIgG1(3A)版本以及人源化10E11和3F5抗体的hIgG4版本：h10E11-hIgG1，h4E4-hIgG1，h4B11-hIgG1，h10E11-hIgG1(3A)，h10E11-hIgG4以及h3F5-hIgG4。氨基酸序列包括h4E4-IgG1[SEQ ID NOs：121(全长重链)和122(全长轻链)]，h10E11-IgG1[(SEQ ID NOs：127及128(全长重链)和131(全长轻链))、h10E11-IgG1(3A)[(SEQ ID NOs：129(全长重链)和131(全长轻链))，h10E11-IgG4[(SEQ ID NOs：130(全长重链)和131(全长轻链))，h4B11-IgG1[(SEQ ID NOs：123(全长重链)和124(全长轻链))和h3F5-IgG1[(SEQ ID NOs：125(全长重链)和126(全长轻链))，如上列表3所示。
人源化4E4、10E11、4B11和3F5抗体的构建和表达：合成编码人源化抗体轻链和重链的DNA，并将其克隆到表达载体pcDNA3.1(Invitrogen，CAT：#V-790)。用100μg每种人源化重链和轻链表达质粒转染Freestyle 293细胞(200mL，10 6/mL)，并培养6天。然后用Protein-G柱(GE Healthcare)纯化上清液中的人源化抗体。
实施例9
使用ELISA和FACS确定人源化抗体与GITR的结合，使用FACS测定GITR配体被阻断的活性，以及基于Jurkat-GITR-NF-κB细胞报告基因测试分析人源化抗体的免疫活化反应。
人源化抗体的产生和纯化后，通过基于ELISA和FACS的结合分析和Jurkat-GITR细胞的GITR-L阻断分析，确定抗体的结合和特异性。所使用的方法与以上实例2和4中所述的方法相似。
在基于ELISA的结合测定中，人源化抗体显示出与GITR的显着结合，如附图1所示，用ELISA方法检测人源化抗GITR抗体与GITR-mFc蛋白结合的EC50；人源化的抗GITR抗体包括h10E11(AV)-hIgG1(3A)，h10E11(AV)-hIgG4，h4E4-hIgG1，h4B11-hIgG1和h3F5-hIgG1。图的上部分显示了在一系列人源化抗体或对照hIgG1浓度范围内的光吸度值。如X轴所示，每种抗体的测试浓度分别为20000ng/mL，2000ng/mL，200ng/mL，20ng/mL，2ng/mL， 0.2ng/mL，0.02ng/mL，0.002ng/mL、0.0002ng/mL和0.00002ng/mL。在图底部的表显示了每种测试抗体所计算的EC50。如图所示，所有人源化抗体显示出与平板包被的GITR-mFc有显著的结合作用，h4B11-hIgG1和h10E11(AV)-hIgG1(3A)具更强的结合力。
图2显示了Jurkat-GITR细胞上基于FACS的与GITR的结合。底部面板提供了每种人源化抗体的EC50。4B11-hIgG1和10E11(AV)-hIgG1是基于FACS分析的主要结合物。人源化GITR抗体包括h10E11(AV)-hIgG1，h10E11(AV)-hIgG1(3A)，h10E11(AV)-hIgG4，h4E4-hIgG1，h4B11-hIgG1，h3F5-hIgG1和阴性对照(人源IgG1)。图的上部分显示了在一系列人源化抗体或对照hIgG1浓度范围内的平均荧光指数值。底部显示了每种测试抗体的计算EC50。结果表明，所有人源化抗体均显示与Jurkat-GITR细胞的显着结合。同时，人源化的h4E4和h10E11(AV)抗体是最主要的粘合剂。如X轴所示，每种抗体的测试浓度分别为20000ng/mL，2000ng/mL，200ng/mL，20ng/mL，2ng/mL，0.2ng/mL，0.02ng/mL和0.002ng/mL。
图3显示了人源化抗体在Jurkat-GITR细胞上GITR-L与GITR的结合阻断。使用的GITR-L浓度为0.25ug/ml)；测试的GITR抗体包括h10E11(AV)-hIgG1，h10E11(AV)-hIgG1(3A)，h10E11(AV)-hIgG4，h4E4-hIgG1，h4B11-hIgG1，h3F5-hIgG1和阴性对照(人源IgG1)。如X轴所示，每种抗体的测试浓度分别为20000ng/mL，4000ng/mL，800ng/mL，160ng/mL，32ng/mL，6.4ng/mL，1.28ng/mL和0.256ng/mL。结果表明，所有人源化GITR抗体对GITR-L与Jurkat-GITR细胞的结合均显示出一定的阻断活性。人源化h10E11(AV)和h3F5抗体具更好的阻断作用。
图4显示了基于293T-GITR-NFkb-luc细胞报告基因测定法的人源化抗GITR抗体的GITR激活活性。测试的抗GITR抗体包括h10E11(AV)-hIgG1(3A)，h10E11(AV)-hIgG1，h4E4-hIgG1，h4B11-hIgG1，h3F5-hIgG1和阴性对照(人源IgG1)。每种抗体的测试浓度分别为20000ng/mL，4000ng/mL，800ng/mL，160ng/mL，32ng/mL，6.4ng/mL，1.28ng/mL和0.256ng/mL。结果表明，所有人源化抗体对293T-GITR-NFkb-luc细胞的荧光素酶报道基因表达均具有较强的激活活性。在测试的人源化抗体之间的活性是相似的。
实施例10
用Jurkat-GITR IL-2释放分析对人源化抗GITR抗体的激活测试
为了测试杂交瘤抗GITR抗体的激活活性，首先通过其对Jurkat-GITR细胞中 GITR信号通路的刺激作用来评估抗体。将Jurkat-GITR，293T-OKT3、293T-CD32A和293T细胞分别在补充有10％FBS的RPMI 1640中培养。将50μl Jurkat-GITR(每孔1x105细胞)和50μl 293T-OKT3(每孔2x104细胞)和50μl293T-CD32A或293T(每孔2x104细胞)细胞添加到96孔板的单个孔中。是否存在稀释的GITR抗体。在37℃下孵育48或72小时后，将96孔板离心并收集上清液以测量IL-2(R&D Systems，目录号DY202)。
图5显示由于人源化抗-GITR抗体活化Jurkat-GITR细胞而没有交联的情况下IL2释放的结果。Jurkat-GITR细胞受293T－OKT3细胞中OKT3一定程度的刺激。测试的抗GITR抗体包括h10E11(AV)-hIgG1(3A)，h10E11(AV)-hIgG1，h4E4-hIgG1，h4B11-hIgG1，h3F5-hIgG1和阴性对照(人源IgG1)。每种抗体的测试浓度分别为20000ng/mL，4000ng/mL，800ng/mL，160ng/mL，32ng/mL，6.4ng/mL，1.28ng/mL和0.256ng/mL。如我们所见，所有的人源化抗体在无需交联的情况下，可以在一定程度上刺激Jurkat-GITR细胞IL2的释放。
图6显示由于人源化抗GITR抗体在存在交联的情况下激活Jurkat-GITR细胞而导致的IL2释放的结果。交联因子为在293T－CD32a细胞中表达的CD32a。Jurkat-GITR细胞受293T－OKT3细胞中OKT3一定程度的刺激。测试的抗GITR抗体包括h10E11(AV)-hIgG1(3A)，h10E11(AV)-hIgG1，h4E4-hIgG1，h4B11-hIgG1，h3F5-hIgG1和阴性对照(人源IgG1)。每种抗体的测试浓度分别为20000ng/mL，4000ng/mL，800ng/mL，160ng/mL，32ng/mL，6.4ng/mL，1.28ng/mL和0.256ng/mL。如我们所见，通过交联，所有人源化抗体对Jurkat-GITR细胞(尤其是h4E4-hIgG1)的IL2释放均表现出更强的刺激活性。
实施例11
人源化抗GITR抗体对CD4+T细胞产生细胞因子的影响
GITR杂交瘤抗体的活性通过其刺激淋巴细胞效应细胞中的GITR信号传导途径的作用来评估。使用补充了10％FBS的RPMI 1640中的CD4+阴性选择试剂盒，从PBMC中纯化人CD4+T细胞。在前一天，将96孔板在室温下用山羊抗人IgG(Jackson，目录号：109-005-008)和山羊抗小鼠IgG(Jackson，目录号：115-006-071)包被过夜。温度。洗涤孔并通过添加200μL封闭缓冲液封闭并在37℃温育1小时。将板用PBS洗涤3次后，在37℃下将40ng/mL OKT3(Ebioscience，目录号：16-0037-85)加入孔中孵育1小时。用PBS洗涤板后，在存在或不存在稀释的GITR抗体的情况下，将100μl CD4+T细胞添加到96孔板上的每个孔中(每孔1x10 5个细胞)。在37℃下温育48或72小时后，将 96孔板离心并收集上清液，以使用ELISA试剂盒(R&D Systems，Cat。Cat)测量IL-2(R&D Systems，目录号：DY202)和IFN-γ的产生编号：DY285)。
图7显示了人源化抗GITR抗体对CD4+T细胞产生IL2的影响。测试的抗GITR抗体包括h10E11(AV)-hIgG1(3A)，h10E11(AV)-hIgG1，h4E4-hIgG1，h4B11-hIgG1，h3F5-hIgG1和阴性对照(人IgG1)。每种抗体的测试浓度分别为20ng/mL，2ng/mL，0.2ng/mL和0.02ng/mL。如图所示，人源化的10E11、4B11和4E4抗体(特别是h4B11-hIgG1)对原代CD4T细胞产生细胞因子IL2具有显著的刺激作用；而人源化3F5抗体在此分析中显示的活性相对较低。
图8显示了人源化抗GITR抗体对CD4+T细胞产生的IFN-γ的影响。测试的抗GITR抗体包括h10E11(AV)-hIgG1(3A)，h10E11(AV)-hIgG1，h4E4-hIgG1，h4B11-hIgG1，h3F5-hIgG1和阴性对照(人IgG1)。每种抗体的测试浓度分别为20ng/mL，2ng/mL，0.2ng/mL和0.02ng/mL。如图所示，人源化10E11、4B11和4E4抗体(特别是h4B11-hIgG1)对原代CD4T细胞产生细胞因子IFN-γ具有显著的刺激作用；而人源化3F5抗体在此分析中显示了较低的活性。结果指定了与图7中IL2产生相似的模式。也就是说，人源化的10E11、4B11和4E4抗体对IL2产生对原代CD4T细胞的刺激具有显著作用。而人源化3F5抗体则显示相对较低的激活活性。
实施例12：人源化4E4、10E11、4B11和3F5抗GITR抗体的Biacore动力学分析
为了表征人源化抗体的结合特性，通过Biacore3000测量了GITR和抗GITR抗体之间的结合动力学，并以1Hz的数据采集速率进行记录。将多克隆兔抗小鼠IgG(GE，BR-1008-38)用10mM pH 5.0乙酸钠稀释并使用胺偶联试剂盒(GE，BR10050)固定在CM5生物传感器芯片的参比和实验流通池上至约15000RU)。在每个循环的开始，将稀释的测试抗体(1.5μg/mL)注入实验流动池中1分钟以捕获。GITR分析物系列的制备方法是：用流动缓冲液将原液稀释至100nM，然后在同一缓冲液中进行2倍系列稀释至0.78nM。将分析物以30μL/分钟的流速连续注入参考和实验流通池3分钟。然后，使运行缓冲液(含0.05％P20的PBS)以30μL/分钟的流速流过10分钟。在每个循环结束时，通过以10μL/min的流速注入10mM pH1.7甘氨酸-HCl缓冲液3分钟，使生物传感器表面再生。对于每次分析物样品注射(即每个循环)，通过从参考表面减去同时记录的响应，然后从单个参考运行缓冲液样品中减去响应，来对从实验生物传感器表面获得的结合响应进行双重参考。通过使用Biaevaluation 4.0软件将整个滴定系列的双参 考传感图拟合到Langmuir模型(1：1)，可以同时确定缔合和解离速率常数(ka和kd)。解离常数KD由确定的速率常数通过关系式KD＝kd/ka计算得出。如表10所示，人源化抗GITR抗体以高亲和力结合人GITR。特别是人性化的4B11和10E11。
表10.GITR-his multi-cycle kinetics test by SPR
Antibody Ka(1/MS) Kd(1/S) KD(M)
Humanized 10E11 4.24E+05 2.93E-04 6.91E-10
Humanized 4E4 3.25E+05 1.23E-03 3.78E-09
Humanized 4B11 1.73E+06 2.57E-04 1.49E-10
Biacore T200 test；Model:kinetics,1:1 binding；KD＝kd/ka
以上实施例的结果表明本发明所述的单克隆抗体或其抗原结合片段，或者包含本发明所述单克隆抗体或其抗原结合片段的单克隆抗体偶联物在制备激活GITR信号传导的药物、激活T淋巴细胞的药物、提高T淋巴细胞表达IL-2和INF-γ的药物、阻断GITR－L与GITR结合的药物、以及在预防和治疗或者辅助治疗肿瘤的药物方面具有良好的应用前景。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进，这些改进也应视为本发明的保护范围。

Claims (9)

  1. 一组抗GITR单克隆抗体或其抗原结合片段，包括重链和轻链，其特征在于，所述重链的CDR1的氨基酸序列选自SEQ ID NO：2,10,18,26,34,42,50,58,66,74,82,90,98和106中的一种；所述重链的CDR2的氨基酸序列选自SEQ ID NO：3,11,19,27,35,43,51,59,67,75,83,91,99和107中的一种；所述重链的CDR3的氨基酸序列选自SEQ ID NO：4,12,20,28,36,44,52,60,68,76,84,92,100和108中的一种；所述轻链的CDR1氨基酸序列选自SEQ ID NO：6,14,22,30,38,46,54,62,70,78,86,94,102和110中的一种；所述轻链的CDR2的氨基酸序列选自SEQ ID NO：7,15,23,31,39,47,55,63,71,79,87,95,103和111中的一种；所述轻链的CDR3的氨基酸序列选自SEQ ID NO：8,16,24,32,40,48,56,64,72,80,88,96,104和112中的一种；其中所述抗原结合片段的重链和轻链包含分别跨越所述抗体的重链和轻链的CDR1到CDR3的氨基酸序列。
  2. 根据权利要求1所述的一组GITR单克隆抗体或其抗原结合片段，其特征在于，所述重链可变区的氨基酸序列选自SEQ ID NO：1,9,17,25,33,41,49,57,65,73,81,89,97和105中的一种；所述轻链可变区的氨基酸序列选自SEQ ID NO：5,13,21,29,37,45,53,61,69,77,85,93,101和109中的一种。
  3. 根据权利要求1所述的一组抗GITR单克隆抗体或其抗原结合片段，其特征在于，所述重链和轻链经过人源化；所述人源化后的重链可变区的氨基酸序列选自SEQ ID NO：113,115,117和119中的一种；所述人源化后的轻链可变区的氨基酸序列选自SEQ ID NO：114,116,118和120中的一种。
  4. 根据权利要求3所述的一组抗GITR单克隆抗体或其抗原结合片段，其特征在于，所述人源化后的重链的全长氨基酸选自SEQ ID NO：121,123，125，127，128，129和130中的一种；所述人源化后的轻链的全长氨基酸选自SEQ ID NO：122,124，126和131中的一种。
  5. 编码如权利要求1-4任一项所述的抗GITR单克隆抗体或其抗原结合片段的核苷酸序列。
  6. 权利要求1-4任一项所述的抗GITR单克隆抗体或其抗原结合片段在制备药物中的用途：所述药物包括但不限于阻断GITR-L与GITR结合的药物、激活T淋巴细胞的药物或者提高T淋巴细胞中IL-2和IFN-表达的药物。
  7. 一种单克隆抗体偶联物，包括单克隆抗体和偶联部分，其特征在于，所述单克隆抗体为权利要求1-4任一项所述的一组抗GITR单克隆抗体或其抗原结合片段，所述偶联部分为选自放射性核素、药物、毒素、细胞因子、细胞因子受体片段、酶、荧光素、和生物素中的一种或多种。
  8. 根据权利要求7所述的一种单克隆抗体偶联物在制备药物中的用途：所述药物包括但不限于阻断GITR-L与GITR结合的药物、激活T淋巴细胞的药物或者提高T淋巴细胞中IL-2和IFN-表达的药物。
  9. 根据权利要求7所述的一种单克隆抗体偶联物在制备预防和/或治疗和/或辅助治疗肿瘤的药物中的用途。
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